Project Summary/Abstract Acute Lung Injury (ALI) and its more severe form Acute Respiratory Distress Syndrome (ARDS) are a common cause of respiratory failure in critically ill patients. All current therapies for ALI/ARDS rely on supportive care to improve clinical outcome. No effective drugs have been developed. There is an urgent need to develop new treatment strategies for ALI/ARDS that are safe, effective, and based on deeper understanding of the mechanisms involved in ALI pathogenesis. We have discovered that MTOR plays a key role in the inflammation associated with ALI and that downregulation of MTOR in lung epithelial cells has the potential to alleviate this inflammation. However, downregulation of MTOR in lung endothelial cells has the opposite effect and exacerbates inflammation. Thus, to translate these findings into a potential treatment, we must reduce MTOR levels and activity selectively in the lung epithelium. We have also recently reported the discovery of disulfide-constrained, cyclic amphipathic peptides (CAPS) that bind to siRNA to form nanocomplexes that can functionally affect intracellular delivery of siRNA cargo to the lung for protein silencing. We hypothesize that CAP-siRNA nanoparticles represent an ideal vector for selective delivery of siRNA to lung epithelial cells by simple aspiration. The overall objective of this proposal is to characterize the mechanism of intracellular siRNA delivery by CAP-siRNA nanoparticles and to optimize MTOR silencing by these particles toward validation of their application as a pharmacologic treatment for ALI. We will utilize a cross-disciplinary strategy to accomplish the stated research objective. The Specific Aims of the proposal are: 1) To characterize the mechanism of translocation for intracellular delivery of siRNA by our recently reported CAPs. 2) To conduct structure-activity studies to optimize the efficiency of intracellular siRNA delivery and gene silencing by CAP- siRNA nanocomplexes. 3) To validate the use of CAP-siRNA nanoparticles for selective knockdown of MTOR in lung epithelial cells in an in vivo model for ALI. The proposed work requires expertise in both the physical and biological sciences. The research team draws on expertise in peptide design, lung biology, and gene therapy. The proposed work will extend existing collaborative relationships between the Nilsson, Dean, and Rahman groups. Accomplishment of the stated research goal will address significant gaps in understanding of the disease etiology of ALI, validate the efficacy of MTOR downregulation for treatment of ALI, and provide peptide/siRNA nanoparticles that facilitate in vivo delivery of MTOR-specific siRNA to the lung. Further, the proposed CAP agents represent a new class of innovative cell-penetrating peptide motif that is simple and inexpensive to produce and that does not require covalent attachment of cargo to promote cell entry. It is anticipated that the proposed CAP-siRNA nanoparticles will be also useful for gene silencing of other lung targets in a range of disorders as well as in other tissues as a platform technology.